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Comparison of protein and peptide fractionation approaches in protein identification and quantification from Saccharomyces cerevisiae

ABSTRACT: Proteomics is a high-throughput technology which has been developed with the aim of investigating the maximum number of proteins in cells in a given experiment. However, protein discovery and data generation vary in depth and coverage when different technical strategies are selected. In this study, three different sample preparation approaches, and peptide or protein fractionation methods, were applied to identify and quantify proteins from log-phase yeast lysate: sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), filter-aided sample preparation coupled with gas phase fractionation (FASP-GPF), and FASP - high pH reversed phase fractionation (HpH). Fractions were initially analyzed and compared using nanoflow liquid chromatography – tandem mass spectrometry (nanoLC-MS/MS) employing data dependent acquisition on a linear ion trap instrument. The number of fractions and analytical replicates was adjusted so that each experiment used a similar amount of mass spectrometric instrument time. A second set of experiments was performed using a Q Exactive Orbitrap instrument, comparing FASP-GPF, SDS-PAGE and FASP-HpH. Compared with results from the linear ion trap mass spectrometer, the use of a Q Exactive Orbitrap mass spectrometer enabled a small increase in protein identifications, and a very large increase in peptide identifications. This shows that the main advantage of using the higher resolution instrument is in increased proteome coverage. A total of 1035, 1357 and 2134 proteins were separately identified by FASP-GPF, SDS-PAGE, and FASP-HpH. Combining results from the Orbitrap experiments, there were a total of 2269 proteins found, with 94% of them identified using the FASP-HpH method. Therefore, the FASP-HpH method is the optimal choice among these approaches, when applied to this type of sample.

INSTRUMENT(S): ion trap, Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

TISSUE(S): Cell Culture

SUBMITTER: Liting Deng

LAB HEAD: Paul A. Haynes

PROVIDER: PXD018854 | Pride | 2020-11-30

REPOSITORIES: Pride

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Comparison of protein and peptide fractionation approaches in protein identification and quantification from Saccharomyces cerevisiae

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